2.1. Experimental details
The pepper plants (Capsicum annuum L.) were used as the experimental material. This work was carried out in a completely randomized design (RCD) with factorial arrangement and was replicated thrice. The main factors were different levels of SA concentrations and drought. This research was carried out an experimental greenhouse at the Faculty of Agriculture, University of Ilam, Iran. The experiment was conducted during four months. The sweet pepper seeds were obtained from the Faculty of Agriculture. Before sowing, seeds sterilized by immersing in 1% sodium hypochlorite for 10 min before being washed with tap water for 1 min. Then, the seeds were sown in plastic pots (23×20 cm). The pots were filled the same amount of sand, garden soil and leaf mold (1: 1: 1) mixture. After that, each pot weighed 7 kg. The soil samples were analyzed to determine different soil properties (Table 1).
The relative humidity (R.H. %) and mean temperature during the growth seasons were 60–70% and 18/ 25 °C (day/ night) respectively. SA was used at three levels, namely 0 (sprayed with distilled water as acontrol), 0.5 and 1mM. The SA was sprayed upper and lower leaves of fourth leaf stage. Tween-20 was used as a surfactant. The first and second spray application of SA was at three days before and two weeks after the drought stress began. In the early stages of plant growth, all pots were watered until field capacity. Accordingly, it lasted approximately four months three days after the foliar spray until sampling. The experiment consisted of three levels of drought stress: full irrigation (control group), 60% and 30% of field capacity, moderate and severe stress respectively. We have not used any fertigation in this experiment. All pots were weighed on a daily basis. These conditions were contended until the end of the investigation.
For the determination of physic-chemical parameters, 27 plants were harvested in the green stage (80% maturity). Leaf samples were harvested by three replicates and each replicate was obtained from three pots, and therefore nine plants existed in each treatment group. The samples were quickly frozen in liquid nitrogen. They were stored at -80 °C.
Growth Parameters
The leaf area (LA) of leaves was measured using an area meter (AM 300 Bioscientific Ltd.UK) fully grown leaves that expanded from the main stem (Phimchan et al. 2012). The leaf chlorophyll index (CHLI) was assessed according to the method described in the study of Wang et al. (2017) from the last fully expanded leaf of sweet pepper by SPAD502. The shoots fresh weight of pepper plants were assessed by a digital analytical balance. The dry weight of shoots were determined after being dried in an oven at 60°C (Ahmed et al. 2014). The roots of the sweet pepper plants were carefully separated and then washed thoroughly with distilled water several times and then measured with a digital analytical balance. Then, they were placed in an oven at 60°C to assess the dry weight of roots (Ahmed et al. 2014). A method by Barranco et al. (2005) was applied for determination of leaf relative water content (RWC). According that, the fresh leaves were cut into 0.1 g disks. The contaminants and residues of samples were removed thoroughly with distilled water. Then, the leaf samples were placed to the test tubes with 10 ml distilled water. The tubes were placed for 30 min at 30°C, subsequently the initial electric conductivity of the solution (Ec1) was measured. For calculating secondary electric conductivity (Ec2), the tubes heated for 15 min at 100°C in bath of water. finely; the total electrical conductivity was calculated using the formula:
EL (%) = (EC1/EC2) × 100.
Fruit physical properties
The morphological characteristics of pepper fruits such as length and diameter of fruit were measured. The measurements of diameter and length of the fruits were taken in maximum width and length. All measurements were by using a Caliber (Thuy and Kenji 2015).
Determine relative electrical conductivity
This test was performed by referring to the method developed by Deshmukh et al. (1991). The fresh leaves Cut (0.1 g) by disk and remove contaminants with distilled water. Then the leaf samples were placed to the test tubes containing 10 ml of distilled water and incubated for 30 min at 30°C. Then the initial electric conductivity of the solution (Ec1) was measured. Subsequently the tubes heated for 15 min at 100°C in a bath of water and the secondary electric conductivity (Ec2) was calculated. Eventually the total electrical conductivity was determined by the following formula:
EL (%) = (EC1/EC2) × 100
Measurement of Non-enzymatic antioxidants
Measurement of AsA content
A modified procedure by Luwe et al. (1993) was estimated for determination AsA content of pepper leaves. Initially, the samples pepper leaves (0.5 g) were poured by mortar and pestle in liquid nitrogen ant then homogenized in cold trichloroacetic acid (TCA, 1% w/v) and centrifuged at 12000×g for 20 min at 4°C. The supernatant was mixed with 50 µL potassium phosphate buffer mixture (0.95 ml, 100 mm, pH 7.0) along with ascorbate oxidase (1 μl of 1 μl-1 unit). Finely, the absorbance was read at 265 nm.
Assessment of Total phenolic content
The total phenol content was estimated per the method of Kahkonen et al. (1999). The leaves of fresh pepper (0.4 g) were homogenized in 4 ml of methanol and 0.1 Mol l-1 HCl mixtures. The mixture was centrifuged at 15000×g for 20 min at 4°C. The supernatant was mixed with distilled water (1:10) and by adding 2 ml diluted solution with 400 μl supernatant. The mixture was then added 1.6 ml of sodium carbonate (Na2CO3 7.5%) to the above solution. The solution was stored in the dark for 30 min and centrifuged at 5000×g for 5 min. Eventually, the absorbance of the mixture was measured at 765 nm. The total phenolic content was estimated by Gallic acid mg ml-1 fresh material.
Antioxidant property
The DPPH was used to determine antioxidant activity of pepper leaves as described by Ab et al. (1998). The fresh leaves (0.2 g) were ground homogeneously with a mortar and pestle with 2 ml of ethanol at 4°C. About half of the solution was blended with a solution consist of 0.5 ml of 100 mm acetate buffer (pH 5.5) and 0.25 ml of 0.5 mM DPPH. The absorbance was recorded at 517 nm after 30 min. The antioxidant properties were calculated using the formula:
Assaying the activity of antioxidant enzymes
Preparation of extract:
The pepper plant leaf was crushed in a mortar containing 50 ml of sodium phosphate buffer and then, centrifuged at 16,000 g at 10°C. The supernatant was to determine antioxidant enzyme activities APX and PPO.
Measurement of polyphenol oxidase activity
Reaction mixture containing 900 μl of catechol and 40 μl of 0.01 M sodium phosphate buffer with pH 6.8 was prepared and then 100 μl of enzyme extract was added. Then, the absorbance was recorded at 400 nm in 25°C for 3 min (Jiang et al. 2002).
Assaying of ascorbate peroxidase enzyme
For this assay, 0.1 ml of enzyme extract was added to a mixture containing 0.1 ml of 0.5 mM ascorbate, 0.2 ml of H2O2 1%, 2.5 ml of 50 mM potassium phosphate buffer and 0.1 mM EDTA and then the absorption was recorded at 290 nm at 25°C (Asada 1992).
Statistical analysis
All statistical analyses were performed by analysis of variance (ANOVA) using SPSS statistical 10 software. Differences between treatments were separated by Duncan’s multiple range tests. The analyses were carried out to determine significant variation between the means at a significance level of P <0.05.